High-content analysis of proteostasis capacity in cellular models of amyotrophic lateral sclerosis (ALS)

Disrupted proteome homeostasis (proteostasis) in amyotrophic lateral sclerosis (ALS) has been a major focus of research in the past two decades. However, the proteostasis processes that become disturbed in ALS are not fully understood. Obtaining more detailed knowledge of proteostasis disruption in association with different ALS-causing mutations will improve our understanding of ALS pathophysiology and may identify novel therapeutic targets and strategies for ALS patients. Here we describe the development and use of a novel high-content analysis (HCA) assay to investigate proteostasis disturbances caused by the expression of several ALS-causing gene variants. This assay involves the use of conformationally-destabilised mutants of firefly luciferase (Fluc) to examine protein folding/re-folding capacity in NSC-34 cells expressing ALS-associated mutations in the genes encoding superoxide dismutase-1 (SOD1A4V) and cyclin F (CCNFS621G). We demonstrate that these Fluc isoforms can be used in high-throughput format to report on reductions in the activity of the chaperone network that result from the expression of SOD1A4V, providing multiplexed information at single-cell resolution. In addition to SOD1A4V and CCNFS621G, NSC-34 models of ALS-associated TDP-43, FUS, UBQLN2, OPTN, VCP and VAPB mutants were generated that could be screened using this assay in future work. For ALS-associated mutant proteins that do cause reductions in protein quality control capacity, such as SOD1A4V, this assay has potential to be applied in drug screening studies to identify candidate compounds that can ameliorate this deficiency.

Table S1.Plasmid combinations used in triple transfections in preparation for HCA microscopy optimisation.Triple-transfected NSC-34 cells were used for preliminary experiments to optimise the parameters used for automated imaging and image analysis using a Cellomics® ArrayScan™ VTI HCS microscope.

Confocal image analysis and inclusion characterisation
To characterise the localisation patterns of each EGFP-/tGFP-and mCherry-tagged ALSassociated protein in transfected NSC-34 cells and ensure that they corresponded with the patterns recorded in the literature, the images acquired by confocal microscopy were manually examined.To inform this characterisation, previous peer-reviewed studies revealing images of post-mortem spinal cord tissue from ALS patients were used to establish the morphology and size range of inclusions positive for mutant SOD1, TDP-43, FUS, VAPB, OPTN, UBQLN2 and VCP.This enabled a size minimum of 2 µm to be established for categorising fluorescent foci as inclusions as opposed to associations of the fluorescent ALS proteins with other cellular structures, granules (e.g stress or transport) or other irrelevant fluorescent debris and artefacts.

IncuCyte® ZOOM live cell imaging and analysis
IncuCyte® ZOOM live cell imaging and analysis was used to carry out two different measures in transfected NSC-34 cells.One of the experiments involved monitoring the relative population growth of transfected cells (EGFP-positive cells) over 72 h.The net cell population growth (rate of cell division -rate of cell death) was used as a measure of cell viability, on the basis that toxicity caused by the overexpression of the mutant ALS gene would impair cellular ability to proliferate and/or result in the loss of cells relative to control cells overexpressing the WT gene or EGFP/mCherry alone.NSC-34 cells were transfected in 6-well plates and were re-plated at 24 h post-transfection at uniform cell density (5 × 10 4 cells/mL) in 200 µL/well DMEM/F-12/FBS into 96-well plates and imaged using the 10× objective lens of an IncuCyte® ZOOM over 72 h.To quantify the numbers of viable transfected cells, a processing definition in the IncuCyte® ZOOM software was optimised to identify and count transfected cells based on a minimum level of EGFP/mCherry fluorescence, and viable cells based on a minimum size.Non-transfected controls were used to account for and exclude background fluorescence and fluorescent artefacts, cell debris and dying cells.To account for variations in transfection efficiency between samples, the numbers of transfected cells were normalised by subtracting the numbers of transfected cells at time zero of the assay (upon replating cells at 24 h post-transfection) from the numbers of transfected cells at each time point, for each replicate.
The second experiment quantified cells containing inclusions using a modification of a saponin-permeabilisation protocol described by Pokrishevsky et al. (2018) [4].Saponin is a mild, cholesterol-chelating detergent that creates pores in the plasma membrane of cells [5].These pores allow soluble intracellular proteins to diffuse out of the cell, while trapping insoluble proteinaceous structures.NSC-34 cells were transfected with each expression construct in quadruplicate in 96-well plates, and after 48 h were imaged using an IncuCyte® ZOOM.These pre-permeabilisation images were used to measure the numbers of cells that were transfected (EGFP-/mCherry-positive) in each well.After imaging, cells were incubated with 0.03% (w/v) saponin in PBS for 10 min at RT to create pores in the cells' plasma membranes.Saponin-treated cells were then imaged again to measure the numbers of cells in which non-diffusable, insoluble EGFP-/mCherry-positive material remained following permeabilisation.The percentage of transfected cells containing insoluble EGFP-/mCherryfusion proteins were then calculated using the following formula: Samples were heated at 95 °C in loading buffer for 5 min prior to SDS-PAGE, in which proteins were resolved on Any-kD™ Criterion™ TGX Stain-Free™ precast gels (BioRad) for 2 h at 100 V. Proteins were subsequently transferred onto PDVF membrane (0.2 µm pore size) for 1 h at 100 V. Membranes were imaged with a GelDoc XR+ imager (BioRad) to obtain a total protein measurement for each sample before incubation with blocking solution containing 5% skim milk powder (w/v) in Tris-Buffered Saline with Tween-20 (TBS-T) for 1 h at room temperature.Blots were probed with the following primary antibodies overnight at 4 °C: For SOD1-EGFP and OPTN-EGFP, rabbit polyclonal antibody to EGFP (ab6556, Abcam, 1:5,000 dilution in blocking buffer); for CCNF-mCherry, rabbit polyclonal antibody to CCNF CC-20 (SC-952, Santa Cruz Biotechnology, 1:2,000 dilution in blocking buffer); for VCP-tGFP, mouse monoclonal antibody to tGFP, horseradish peroxidase (HRP)-conjugated (TA150043, OriGene, 1:5,000 dilution in blocking buffer).Blots were washed four times (10 min) in TBS-T, and incubated with goat anti-rabbit HRP-conjugated secondary antibody (A16104, Invitrogen, 1:5,000 dilution in blocking buffer) for 1 h at room temperature.Bands were visualised by briefly incubating the membranes in SuperSignal™ West Dura Chemiluminescent Substrate according to the manufacturer's instructions (ThermoFisher Scientific) and imaged using an Amersham 600RB Imager (GE Healthcare).Relative fusion protein levels for each sample were quantified using ImageJ gel analysis tools and normalised to their respective total protein measurement, to account for protein loading variability.(ii) To ensure that cells with particularly low expression of GFP-fusion genes were not included for analysis, GFP fluorescence intensity limits for CircAvgIntenCh2 were set to a minimum intensity of 100 RFU and a maximum intensity of 3241 RFU.(iii) To detect and analyse fluorescent foci corresponding to protein inclusions, as established using confocal microscopy, a spot area minimum of 2 µm 2 and maximum of 150 µm 2 was set.As inclusions are accumulations of high concentrations of proteins, the concentrated fluorescence of GFP-tagged proteins in inclusions allows them to be detected based on high fluorescence intensity.A spot average fluorescence intensity minimum of 150 RFU was found to detect appropriate foci corresponding to GFP-positive inclusions.(D) Channel 3 objects were identified using the same mask as Channel 2, after establishing the fluorescence intensity threshold using the Isodata method with an offset value of 6. (iii) Quantitative assessment of (i) fusion protein levels (bands indicated by arrow heads) were analysed compared to (ii) total protein using ImageJ software.Graphs represent the mean ± SEM from n = 3 biological repeats.Student's t tests or one-Way ANOVA followed by Tukey's Multiple Comparison Test were used to compare differences between means, which were not significant.Non-specific antibodybinding resulted in the presence of additional bands in each blot.Bands greater than 250 kDa in (B, i) may represent large complexes containing CCNF [6].Immunoblot analysis in (C) includes samples from a model not described in this study, OPTN Q398X -EGFP, as these samples were run on the same gel as the other OPTN cellular models, and the complete, unmodified blot is shown for transparency.

Toxicity of mutant UBQLN2, OPTN, VAPB and VCP
Assaying cell population growth using live cell imaging over 72 h, expression of UBQLN2 P497H -tGFP and of UBQLN2 P525S -tGFP was found to cause reductions in cell population growth rates compared to the expression of UBQLN2 WT -tGFP (UBQLN2 P497H -tGFP, p < 0.0001; UBQLN2 P525S -tGFP, p < 0.001) (Figure S3, b).Interestingly, expression of the UBQLN2 P497H -tGFP mutant was observed to cause greater inhibition of cell population growth compared to the UBQLN2 P525S -tGFP mutant, with UBQLN2 P497H -tGFP cells exhibiting slower population growth rates (p = 0.0275) and dropping down to significantly lower numbers by 72 h post-transfection (p = 0.0264) than UBQLN2 P525S -tGFP cells (Figure S3, c, i).In NSC-34 cells expressing the VAPB-tGFP constructs, a clear reduction was observed in the population growth of cells expressing VAPB P56S -tGFP compared to cells expressing VAPB WT -tGFP (Figure S6).Although numbers of transfected cells were low due to low transfection efficiency, total cell density was high, increasing from ~70% to ~95% over the 72 h imaging period (data not shown).There was thus no potential effect of low total cell density on the proliferation or loss of the VAPB-tGFP-expressing cells.Comparison of the numbers of VAPB-tGFP cells in the first 20 h of imaging confirmed that VAPB P56S -tGFP significantly reduced the population growth rate of cells (p = 0.0007) (Figure S6, b).The numbers of tGFP-positive cells expressing VAPB P56S -tGFP at 48 h post-replating were significantly lower than cells expressing VAPB WT -EGFP (p = 0.0007) (Figure S6, c, i), further highlighting the toxicity caused by expression of the P56S mutation.The expression of OPTN E478G -EGFP reduced the population growth rate of transfected cells compared to cells expressing OPTN WT -EGFP (Figure S7).In the first 20 h of imaging, the population growth rate of OPTN E478G -EGFP-expressing cells was significantly slower than that of OPTN WT -EGFP expressing cells (p = 0.0011) (Figure S7, b).Comparison of the numbers of transfected cells at 48 h post-replating showed that there were significantly more viable OPTN WT -EGFP-expressing cells than of OPTN E478G -EGFP-expressing cells (p = 0.0011) (Figure S7, c, i), demonstrating that overexpression of the E478G mutant caused considerable toxicity in NSC-34 cells.Assaying the numbers of NSC-34 cells expressing the VCP-tGFP constructs over 72 h, it was observed that both mutants significantly reduced the population growth rate of transfected cells relative to VCP WT -tGFP (Figure S8).The population growth rate of cells expressing VCP R191Q -tGFP was notably slower than that of cells expressing VCP R159H -tGFP (p = 0.0019), and 48 h after replating cells the numbers of viable VCP R191Q -tGFP-expressing cells were much lower than the numbers of VCP R159H -tGFP cells (p = 0.0019) (Figure S8, b and c, i).

Characterisation of mutant UBQLN2, OPTN, VAPB and VCP solubility, localisation and aggregation
The saponin-permeabilisation assay was used to examine the mobility and solubility of UBQLN2 P497H -tGFP, UBQLN2 P525S -tGFP, OPTN E478G -EGFP, VAPB P56S -tGFP, VCP R159H -tGFP and VCP R191Q -tGFP in NSC-34 cells.In cells expressing the UBQLN2-tGFP constructs it was found that UBQLN2 WT -tGFP had a degree of immobility in cells, with 37% of transfected cells quantified to contain tGFP-positive protein following permeabilisation of their plasma membranes (Figure S9, a, ii).However, the two UBQLN2 mutants, UBQLN2 P497H -tGFP and UBQLN2 P525S -tGFP, remained inside significantly more cells following permeabilisation; 65.1% Unfortunately, transfections with the VAPB-tGFP constructs for the IncuCyte-based assays resulted in considerably low transfection efficiencies, as can be seen in the low numbers of tGFP-positive transfected cells in Figure S6.The numbers of tGFP-positive cells at 48 h posttransfection detected pre-and post-permeabilisation with saponin for VAPB P56S -tGFP were thus too low for analysis.This saponin-treatment experiment also may not have been appropriate for VAPB, as it is an integral membrane protein and thus in its native localisation in cells is immobile and unable to freely diffuse out of permeabilised cells.

Figure S1 .
Figure S1.Schematic of Cellomics® ArrayScan™ VTI High Content Screening (HCS) image processing and analysis optimisation using Thermo Scientific™ HCS Studio™ software.To analyse the fluorescence intensity of EGFP-/tGFP-and tdTomato (tdT)/mCherry-fusion proteins and quantify protein inclusions containing EGFP-/tGFP-fusion proteins in NSC-34 cells, an image analysis algorithm designed to analyse fluorescent foci in cells, termed the Cellomics® Spot Detector BioApplication, was optimised using the Thermo Scientific™ HCS Studio™ software.Optimisation was carried out using images of NSC-34 cells triple-transfected to express H2B-ECFP, either SOD1 WT -EGFP, SOD1 A4V -EGFP, TDP-43 WT -tGFP TDP-43 M337V -tGFP, FUS WT -tGFP, FUS R495X -tGFP, FUS R521G -tGFP or EGFP alone and mCherry alone.Cells were imaged at 48 h post-transfection using the 20× objective lens of a Cellomics® ArrayScan™ VTI HCS microscope.(A) Raw images from Channels 1 (H2B-ECFP), 2 (EGFP-/tGFP-fusion proteins) and 3 (tdTomato/mCherry-fusion proteins) were first pre-processed to (i) remove background fluorescence using a low-pass filtration method (local background around each pixel is calculated, with the radius of the area sampled adjusted as determined by the user), (ii) exclude cells positioned on the border of each image from analysis and (iii) distinguish individual cells; 'object' segmentation.An object segmentation method based on fluorescence intensity was used, which separates objects/cells that are touching based on fluorescence intensity peaks of each pixel.Cells expressing H2B-ECFP in their nucleus exhibit a single, high intensity peak localised in the nucleus.Setting this parameter, ObjectSegmentationCh1, to a negative value sets the segmentation method to the Intensity method.The absolute value selected governs the minimum relative height of the intensity peak to be used for segmentation.A value of -19 relative fluorescence units (RFU) was found to optimally separate touching fluorescent cells.(iv) Channel 1 images were additionally smoothed (blurred) to help reduce fluorescent noise that could lead to the false inclusion of image artefacts in subsequent analyses.(B) Biological 'objects', in this case cells, were identified using nuclear-localised H2B-ECFP fluorescence in Channel 1 images.(i) For detection of ECFP-fluorescent nuclei, a fluorescence intensity threshold was set using the Isodata method, which derives the threshold from the distribution of pixel intensities in each image.(ii) To select viable transfected cells for analysis and exclude image artefacts, dead cells and cell debris, cells were selected based on the size and fluorescence intensity of their ECFP-fluorescent nuclei.(C) The relevant measures for GFP fluorescence intensity and fluorescent foci were measured in Channel 2 (i) within a circular analysis mask that expanded the mask derived in Channel 1 by 7 µm.The green circular mask indicates cells selected for analysis, while yellow masks indicate fluorescent foci/'spots' selected for analysis.A fluorescence intensity threshold was set for Channel 2 using the Isodata method.An offset value of 1.11 enabled the image processing algorithm to correctly identify cells expressing the GFP-fusion proteins.(ii) To ensure that cells with particularly low expression of GFP-fusion genes were not included for analysis, GFP fluorescence intensity limits for CircAvgIntenCh2 were set to a minimum intensity of 100 RFU and a maximum intensity of 3241 RFU.(iii) To detect and analyse fluorescent foci corresponding to protein inclusions, as established using confocal microscopy, a spot area minimum of 2 µm 2 and maximum of 150 µm 2 was set.As inclusions are accumulations of high concentrations of proteins, the concentrated fluorescence of GFP-tagged proteins in inclusions allows them to be detected based on high fluorescence intensity.A spot average fluorescence intensity minimum of 150 RFU was found to detect appropriate foci corresponding to GFP-positive inclusions.(D) Channel 3 objects were identified using the same mask as Channel 2, after establishing the fluorescence intensity threshold using the Isodata method with an offset value of 6.

Figure S3 .
Figure S3.Nuclear TDP-43 is not released from cells following saponin-permeabilisation.NSC-34 cells were transiently transfected with TDP-43 WT -tGFP or TDP-43 M337V -tGFP.After 48 h, transfected cells were imaged on a Leica TCS SP5 II confocal microscope, followed by incubation with 0.03% (w/v) saponin in PBS for 15 min at room temperature, during which cells were imaged at 5, 10 and 15 min.Representative confocal images of cells prior to saponin-permeabilisation and throughout the incubation period, showing that nuclear TDP-43 was not released from cells following saponin-permeabilisation.White arrow heads indicate cytoplasmic inclusions formed by TDP-43 M337V -tGFP.Scale bars represent 10 µm.

Figure S5 .
Figure S5.ALS-associated UBQLN2 P497H and UBQLN2 P525S cause toxicity in NSC-34 cells.NSC-34 cells were transiently transfected with UBQLN2 WT -tGFP, UBQLN2 P497H -tGFP or UBQLN2 P525S -tGFP and imaged in an IncuCyte® ZOOM over 72 h.Graphs represent the mean ± SEM of (A) numbers of tGFP-positive transfected cells over 72 h, (B) population growth rates of transfected cells and (C, i) numbers of transfected cells at 48 h postreplating, in triplicate wells of cells.(C, ii) Representative IncuCyte images of NSC-34 cells at 48 h post-replating, from which the graph in C, i, was derived.Scale bar represents 150 μm.Differences between the means were determined using one-Way ANOVA followed by Tukey's Multiple Comparison Test.* indicates p < 0.05, *** indicates p < 0.001, **** indicates p < 0.0001.

Figure S6 .
Figure S6.ALS-associated VAPB P56S causes toxicity in NSC-34 cells.NSC-34 cells were transiently transfected with VAPB WT -tGFP or VAPB P56S -tGFP and imaged in an IncuCyte® ZOOM over 72 h.Graphs represent the mean ± SEM of (A) numbers of tGFP-positive transfected cells over 72 h, (B) population growth rates of transfected cells and (C, i) numbers of transfected cells at 48 h post-replating, in triplicate wells of cells.(C, ii) Representative IncuCyte images of NSC-34 cells at 48 h post-replating, from which the graph in C, i, was derived.Scale bar represents 150 μm.Differences between the means were determined using Student's t tests.*** indicates p < 0.001.

Figure S7 .
Figure S7.ALS-associated OPTN E478G causes toxicity in NSC-34 cells.NSC-34 cells were transiently transfected with OPTN WT -EGFP or OPTN E478G -EGFP and imaged in an IncuCyte® ZOOM over 72 h.Graphs represent the mean ± SEM of (A) numbers of EGFP-positive transfected cells over 72 h, (B) population growth rates of transfected cells and (C, i) numbers of transfected cells at 48 h post-replating, in triplicate wells of cells.(C, ii) Representative IncuCyte images of NSC-34 cells at 48 h post-replating, from which the graph in C, i, was derived.Scale bar represents 150 μm.Differences between the means were determined using Student's t tests.** indicates p < 0.01.

Figure S8 .
Figure S8.ALS-associated VCP R159H and VCP R191Q cause toxicity in NSC-34 cells.NSC-34 cells were transiently transfected with VCP WT -tGFP, VCP R159H -tGFP or VCP R191Q -tGFP and imaged in an IncuCyte® ZOOM over 72 h.Graphs represent the mean ± SEM of (A) numbers of tGFP-positive transfected cells over 72 h, (B) population growth rates of transfected cells and (C, i) numbers of transfected cells at 48 h post-replating, in triplicate wells of cells.(C, ii) Representative IncuCyte images of NSC-34 cells at 48 h post-replating, from which the graph in C, i, was derived.Scale bar represents 150 μm.Differences between the means were determined using one-Way ANOVA followed by Tukey's Multiple Comparison Test.** indicates p < 0.01, *** indicates p < 0.001 and **** indicates p < 0.0001.

Figure S9 .
Figure S9.Analysis of the release of EGFP-/tGFP-fusion mutant UBQLN2, OPTN and VCP from NSC-34 cells following saponin-permeabilisation.NSC-34 cells were transiently transfected with (A) UBQLN2WT-tGFP, UBQLN2P497H-tGFP or UBQLN2P525S-tGFP, (B) OPTNWT-EGFP or OPTNE478G-EGFP or (C) VCPWT-tGFP, VCPR159H-tGFP or VCPR191Q-tGFP.After 48 h, cells were (i) fixed, nuclei stained with Hoechst and imaged using a Leica TCS SP5 II confocal microscope or (ii and iii) imaged on an IncuCyte® ZOOM, followed by incubation with 0.03% (w/v) saponin in PBS for 10 min at room temperature, before being imaged again on the IncuCyte.(ii) Cells were transfected in quadruplicate, and the data presented in each graph is the mean ± SEM of the percentage of transfected NSC-34 cells containing insoluble EGFP-/tGFP-positive protein following permeabilisation with saponin.Differences between the means were determined using either Student's t tests